Staphylococcus aureus uses quorum sensing and nutrient availability to control the expression of agr-regulated virulence factors. Quorum sensing is mediated by autoinducing peptide (AIP), which at high concentration, reduces expression of surface attachment proteins (coa, fnbpA), and increases expression of exotoxins (lukS) and proteases (splA). Nutrient availability can be sensed through the saeS/saeR system. Low nutrients increase expression of saeR, which augments expression of coa and fnbpA distinct from AIP. The formation of spatial structure, such as biofilms, can alter quorum sensing and nutrient acquisition. In natural environments, biofilms encounter forces that may alter their spatial structure. This may impact quorum sensing and/or nutrient acquisition, and thus affect the expression of agr-regulated virulence factors. However, this has not been studied. We show that periodically disturbing biofilms composed of S. aureus using a physical force affects the expression of agr-regulated virulence factors. In nutrient-poor environments, disturbance increased the expression of coa, fnbpA, lukS, and splA. Disturbance into a nutrient-rich environment at low or high disturbance amplitudes moderately reduced expression of coa and fnbpA but increased expression of lukS and splA. Interestingly, at an intermediate amplitude, the overall expression of agr-regulated virulence factors was the lowest; expression of lukS and splA remained unchanged relative to an undisturbed biofilm while expression of coa and fnbpA significantly decreased. We hypothesize that these changes are a result of disturbance-driven changes in access to AIP and nutrients. Our results may allow the identification of environments where virulence is enhanced, or reduced, owing to disturbance.